A maraviroc-resistant HIV-1 with narrow cross-resistance to other CCR5 antagonists depends on both N-terminal and extracellular loop domains of drug-bound CCR5

Abstract

CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile.

FIG. 1.

Viruses from patient 6061 evolved resistance to maraviroc but remained sensitive to most other CCR5 antagonists and enfuvirtide. Viruses pseudotyped with Envs cloned from patient 6061 at days 1, 168, and 224 on maraviroc therapy were used to infect NP2/CD4/CCR5 cells in the absence or presence of various concentrations of maraviroc (A), aplaviroc (B), vicriviroc (C), CMPD-167 (D), TAK779 (E), and enfuvirtide (F), and maximal percent inhibition was calculated. Viral Envs cloned after 168 and 224 days on maraviroc therapy are resistant to maraviroc and partially cross-resistant to TAK779 but remain sensitive to all other CCR5 antagonists and the fusion inhibitor enfuvirtide. Data represent results from six independent experiments ± standard errors of the mean.

FIG. 2.

Viral resistance to maraviroc is dependent upon residues in the V3 loop of gp120 and is modulated by the V4 loop. (A) Chimeric viruses were created between the maraviroc-sensitive clone S2 and the maraviroc-resistant clone R3. The V3 loop is required for resistance to maraviroc, while the V4 loop can modulate the magnitude of resistance conferred by V3 but does not impart resistance by itself. (B) Point mutations present in the V3 loop of clone S2 were introduced into clone R3 to determine which residues were critical for maraviroc resistance. The H308P revertant greatly increased the sensitivity of clone R3 to maraviroc, while the H320T and V322aI mutations had more modest effects. (C) The V3 loop from resistant clone R3 was introduced into the ADA, BaL, and SF162 Envs. Transferring the V3 loop conferred a significant degree of maraviroc resistance to these heterologous Envs. Data are representative of results from four independent experiments ± standard errors of the mean.

FIG. 3.

Cloned Envs from patient 6061 utilize CCR5 very efficiently, even in the presence of maraviroc. (A) Infection of pseudotyped viruses bearing Env clone S2 on the Affinofile cell line in the absence of drug. Clone S2 is sensitive to low levels of CD4 but can utilize low levels of CCR5 very efficiently. Infection is normalized to 100% at the highest CD4 and CCR5 levels. Pseudoviruses bearing Env clone S2 were completely inhibited by maraviroc (not shown). Infection of Affinofile cells with pseudotypes bearing maraviroc-resistant Env R3 in the absence (B) or presence (C) of 10 μM maraviroc. Clone R3 is also sensitive to low levels of CD4 but can infect cells bearing very low CCR5 levels both in the absence and presence of maraviroc. (D) Maximal percent inhibition of clone R3 by 10 μM maraviroc at different CCR5 densities was calculated from the data in panels B and C. Inhibition of clone R3 by maraviroc was not strongly impacted by cell CCR5 levels. Data are representative of results from four independent experiments ± standard errors of the mean.

FIG. 4.

Maraviroc-resistant clone R3 is more sensitive to sCD4 and b12 and more resistant to 2G12 than sensitive clone S2. Pseudotyped viruses bearing Env clones S2 and R3 were preincubated with various concentrations of sCD4 (A), b12 (B), 17b (C), 2G12 (D), 2F5 (E), and HIV Ig (F) for 1 h and spinoculated with NP2/CD4/CCR5 cells. The maraviroc-resistant clone R3 is significantly more sensitive to sCD4 and b12 and more resistant to 2G12 than the maraviroc-sensitive clone S2. Data are representative of results from three independent experiments ± standard errors of the mean.

FIG. 5.

CCR5 antagonists disrupt the extracellular loops of CCR5 but have minor effects upon the N terminus. NP2/CD4/CCR5 cells were incubated with medium alone or medium containing saturating concentrations of maraviroc (MVC), aplaviroc (APL), vicriviroc (VVC), CMPD-167, or TAK779. NP2/CD4/CXCR4 cells were utilized as controls. Cells were probed with the N terminus-specific anti-CCR5 monoclonal antibodies (MAbs) 3A9, CTC5, and CTC8 or the extracellular loop (ECL)-specific anti-CCR5 MAbs 2D7, 45523, 45529, 45531, or 45549. CCR5 antagonists had no effect on the binding of N terminus-specific MAbs 3A9 and CTC5 and minor effects on CTC8 binding. In contrast, CCR5 antagonists caused significantly reduced binding of the ECL-specific MAbs 45523, 45529, and 45531 and minor disruption of 2D7 and 45549 binding. Different CCR5 antagonists reduced binding by ECL-specific MAbs to various degrees, suggesting that these drugs induce inhibitor-specific alterations to the ECLs. Data are representative of results from three independent experiments.

FIG. 6.

Model of cross-resistance of Envs to CCR5 antagonists. (A) CCR5 antagonist-resistant viruses that only require the N terminus for binding in the presence of drug recognize a region of the coreceptor that is relatively unaffected by different drugs. As a consequence, these viruses can utilize wild-type CCR5 or CCR5 bound to a variety of different CCR5 antagonists, resulting in broad cross-resistance. In contrast, other resistant viruses require both the N terminus and the extracellular loops (ECLs) of CCR5. Unlike the N terminus, the ECLs of CCR5 are disrupted in an inhibitor-specific manner. Thus, the maraviroc (MVC)-resistant virus can recognize MVC-bound CCR5 but not aplaviroc (APL)- or vicriviroc (VVC)-bound conformations, resulting in narrow cross-resistance. (B) Effect of N-terminal ECL1, ECL2, and ECL3 mutations on drug resistance of the APL-resistant Env Pre5.2 (data from reference 48) and the MVC-resistant Env R3 (data from Table 2). Mutations are shaded according to the degree to which they eliminate drug resistance, with those abolishing resistance shaded red and those having no effect on drug resistance shaded yellow. Data are representative of results from three independent experiments ± standard errors of the mean.